Historically, rodent models of liver carcinogenesis have heavily relied upon administering exogenous toxic agents that induce injury and/or carcinogenesis, in part due to the liver conveniently being the central organ for toxin metabolism. In addition to being extremely practical immunocompetent in vivo models, these agents can also recapitulate features of premalignant and malignant disease, which may not accompany other models that depend strictly on genetic perturbations, such as ductular reactions or fibrosis. A wide array of liver injury agents exist.2,3 Those that have been utilized to study cholangiocarcinoma (CCA) in particular are listed in Table 1. Briefly, the nitrosamines, diethylnitrosamine and dimethylnitrosamine, and thioacetamide (TAA) have both been used as carcinogens, as they can reliably lead to cancer without additional agents or engineered mutations.13,16–18 It is important to note that hepatocellular carcinoma (HCC) may be the dominant tumor type arising in carcinogenic models. Most often, an additional oncogenic event, such as liver fluke infection or biliary-specific deletion of a tumor suppressor, is needed to promote iCCA development.17,18 Although such extreme carcinogenic injury is not frequently recognized epidemiologically among human iCCA, tumors arising in this context are likely more genetically diverse and, therefore, can be useful for study when the genetic context is of minimal importance. Carbon tetrachloride (CCl4), a less severe agent, induces liver fibrosis and cirrhosis, relevant features of human CCA, and, in a Tp53-null setting, promotes iCCA development.19 Finally, bile duct ligation (BDL) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) are used to induce cholestatic injury and ductular reactions, a common feature of liver fluke infections and early iCCA development.13,20–22 When deciding which chemical agents to use, one must consider both the context in which the toxin is to be used and which aspects of injury are most important for the study. For example, if a defined genetic context is desired, a carcinogenic injury model that has an unpredictable genetic progression should be discouraged. In other studies, groups may want to induce a specific feature of liver injury for which a chemical or surgical model is best suited. For example, in an extended experiment assessing the impact of ductular reactions on iCCA development, a flexible dietary model like DDC may be preferable to a surgical model BDL, which is more suitable for short-term experiments. These various models continue to play a key role in the study of liver-related malignancies, and in particular, integrating them with other advanced genetic tools, as addressed in the following sections, has shed much light on the development and biology of iCCA.

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(To view a larger version of Table 1, click here.)


Conditional GEMMs that utilize Cre-Lox technology have been one of the most powerful tools available to understand the development and progression of cancer.23 In this review, we outline how this technology has been utilized to study the liver and liver-related malignancies and highlight a number of important considerations (Table 2). Cre-Lox technology is a conditional system that uses a Cre-recombinase allele to excise DNA between two LoxP sites oriented in the same direction. Depending on the location of LoxP sites, it can be used to both inactivate and activate genes. Engineered alleles for oncogenic Kras and Tp53 provide examples and have been used cooperatively in models of iCCA. The Trp53flox allele has LoxP sites engineered in introns 1 and 10, leading to deletion of exons 2–10 upon Cre-mediated recombination, thereby inactivating Tp53.36 Alternatively, an oncogenic allele can be rendered conditional by inserting a Stop codon flanked by a LoxP site upstream. The KrasLSL-G12D allele is composed of the constitutively active Kras mutant which in the absence of Cre-recombinase is not transcribed due to the upstream Lox-Stop-Lox (LSL) element; however, upon Cre-mediated recombination, the Stop codon is excised and the oncogenic allele is expressed.37

(To view a larger version of Table 2, click here.)

Tissue specificity and temporal control can be achieved with this system by placing the Cre under the control of a cell- or tissue-specific promoter, thereby generating LoxP site recombination exclusively in that lineage (Figure 1). Alternatively, viruses that have tissue-specific tropisms can be used to deliver Cre-recombinase. Adeno-associated virus 8 (AAV8) is a notable example that has a hepatocyte-specific tropism and is therefore frequently used to target hepatocytes while excluding biliary epithelial cells (BECs).38 In addition, the CreERT system, a fusion of Cre and the tamoxifen-inducible domain of the estrogen receptor (ER), enables spatiotemporal control, which has been crucial for the generation of BEC-specific Cre alleles and manipulating genes in the adult mouse that would otherwise be necessary for normal development.21,39